The goals of this project are to define the mechanisms and extent of nucleotide expression repair (NER) inhibition at telomeres, and the impact of unrepaired DNA lesions on telomere structure and function. Telomeres at chromosome ends are essential for genome stability and sustained cell proliferation. Telomeric DNA is highly susceptible to photoproduct formation caused by ultraviolet (UV) light, which are removed by NER in the bulk genome. This proposal will test the hypothesis that telomere binding proteins prevent DNA lesion removal at telomeres by inhibiting the enzymatic activities of NER enzymes. Our preliminary studies show that photoproducts induce telomere loss and aberrations by interfering with telomere replication, which is consistent with a deficiency in lesion removal at telomeres. We further show that a telomeric protein inhibits the catalytic activity of a nuclease required for NER in vitro. Aim 1 will compare endpoints of telomeric damage and dysfunction in UVC irradiated NER proficient- and deficient- cells to establish how photoproducts impact individual telomeres. We will measure photoproduct repair rates in telomeres, compared to the bulk genome, using an innovative assay that quantifies photoproducts in telomeres isolated from UVC exposed human cells. Aim 2 will test for recruitment of key NER proteins to damaged telomeric regions, compared with non-telomeric regions, in cell nuclei using fluorescent protein tags and live cell imaging. Laser micro-irradiatin will be used to generate photoproducts and bulky adducts at define regions in the cell nucleus. Aim 3 will examine how telomeric proteins modulate various enzymatic steps in the NER process. NER will be examined in vitro using cell extracts on defined telomeric and non-telomeric substrates in the presence of individual telomeric proteins or the complete telomeric protein complex. NER is required for removing a wide variety of DNA lesions generated by environmental genotoxicants and anti-cancer drugs. This project will fill a significant void in our understanding of how telomeres evade NER, and how unrepaired DNA lesions alter telomere structure and function. This knowledge will be highly valuable for developing new strategies that 1) preserve telomeres to mitigate the effects of environmental genotoxicant exposures or conversely, that 2) inhibit global genome NER to sensitize malignant cells for killing by anti-cancer genotoxic drugs.